1. Field of the Invention
The present invention relates to a method for measuring polyamines in erythrocytes, a diagnostic method and a kit for measuring polyamines in erythrocytes. More specifically, the invention relates to a method for measuring polyamines in erythrocytes in which amount(s) of spermine (hereinafter referred to also as “Spm”) and/or spermidine (hereinafter referred to also as “Spd”) in erythrocytes can selectively be measured for studies or clinical purposes, a diagnostic method in which disease conditions or physiological conditions of specimens can be diagnosed using the results of the measuring method, and a kit for measuring polyamines in erythrocytes which can be used in such a measuring method or diagnostic method.
2. Description of the Related Art
[Study of In-Vivo Polyamines]
Polyamines inherently include basic hydrocarbon compounds having two or more primary amino groups or secondary amino groups. However, as polyamines found in vivo, putrescine, cadaverine, spermine, spermidine and the like have been known. Of these, spermine is N,N′-bis(3-aminopropyl)-1,4-diaminobutane [NH2(CH2)3NH(CH2)4NH(CH2)3NH2]. Spermidine is N-(3-aminopropyl)-1,4-diaminobutane [NH2(CH2)3NH(CH2)4NH2]. When merely “polyamines” are referred to, they indicate these polyamines found in vivo.
Polyamines are present in large quantities in cells of all organisms ranging from viruses to humans. It has been known that although polyamines are compounds having very simple structures, they interact with various acidic high-molecular materials to exhibit various physiological activities. In recent years, polyamines have attracted much interest in relation to proliferation and differentiation of cells in particular. For example, their relations to DNA synthesis, RNA synthesis, protein synthesis and the like have been studied in the wide range using a regenerated liver and the like. A knowledge which can be applied at clinical levels has been increased.
For example, Dykstra et al. have reported in the following “Literature 1” that after partial excision of the rat liver, the concentration of spermidine is increased for 24 hours, while the concentration of spermine is not increased. In this case, an accumulation rate of the polyamine is nearly the same as an increase rate of RNA synthesis in the regenerated liver, and incorporation of putrescine intravenously injected and synthesis of spermidine are increased within 2 hours after operation of the liver excision. The polyamines are measured using an extract of the rat liver.
Literature 1: William G. Dykstra, Jr.; Edward J. Herbst “Spermidine in Regenerating Liver: Relation to Rapid Synthesis of Ribonucleic Acid” Science, New Series, Vol. 149, No. 3682 (Jul. 23, 1965), 428–429
Russell et al. have reported in the following “Literature 2” that abnormally high polyamine values in urine were identified in patients with various cancers. After this report, a large number of reports have been made on analysis of polyamines in urine, serum, erythrocytes and the like as an index of effects in a diagnostic method, a chemotherapy and the like of malignant tumors.
Literature 2: Diane Russell, Solomon H. Snyder “Amine Synthesis in Rapidly Growing Tissues: Ornithine Decarboxylase Activity in Regenerating Rat Liver, Chick Embryo, and various Tumors” Proc. Natl. Acad. Sci. U.S.A., 1968;60:1420–1427
[Method for Measuring Polyamines]
Under these circumstances, various methods for measuring polyamines in biological samples have been proposed, and some of them have already been put to practical use or are being put to practical use. One of the typical methods is an HPLC method (high-performance liquid chromatography method), which has been utilized for measuring polyamines in blood or polyamines in urine. However, this method is, as well known, considered to be intricate in processing and hardly spread routinely as a common clinical examination method.
Meanwhile, Kubota et al. exploited a method for measuring polyamines in urine using enzymes. This measuring method has been reported in “Igaku no Ayumi” 124(1), 22–24, 1983 and the like. Since this method was exploited, the measurement of total polyamines in urine has been routinized as auxiliary diagnosis of all malignant tumors or as an auxiliary method for estimating therapeutic effects in surgical treatment, administration of antitumor agents, radiotherapy and the like on malignant tumors. Kits for measuring polyamines in urine have been also on the market.
Furthermore, Isobe et al. have reported in the following “Literature 3” the measurement of total polyamines in blood by an enzymatic method.
Literature 3: Kimiyasu Isobe, Hideaki Yamada, Yasuko Soejima, Shogo Otsuji, “A Rapid Enzymatic Assay for Total Blood Polyamines” Clin Biochem, Vol. 20, pp. 157–161, 1987
[Enzymes Used in an Enzymatic Method]
Polyamine oxidases are used in the Kubota et al method or the Isobe et al method. Okada et al have reported properties and the like of various polyamine oxidases in the following “Literature 4”. According to this report, polyamine oxidases are roughly classified into those having a wide-range substrate specificity including a specificity to monoamines and those which specifically react with polyamines including diamines. As an example of enzymes which specifically react with polyamines, an oat-derived polyamine oxidase having a substrate specificity to spermine and spermidine only has been introduced.
Literature 4: Masato Okada, Seiichi Kawashima, Kazutomo Imabori, “Polyamine Sanka Koso” Tanpakushitsu Kakusan Koso vol. 26, No. 9 (1981)
According to the following “Literature 5” of Smith, it has been reported that a barley-derived polyamine oxidase allows specific enzymolysis of spermine with fixed pH.
Literature 5: T. A. Smith “Purification and Properties of the Polyamine Oxidase of Barley Plants” Phytochemistry, 1972, Vol. 11, pp. 899 to 910
Incidentally, a method for measuring polyamines by the foregoing Kubota et al enzymatic method is not said to be simple enough because polyamines in urine are in a conjugated form and therefore require pretreatment of hydrolysis thereof into a free form. Further, when urine is used, a secretory amount of urine water varies with a difference in water intake or action state, a difference in season and the like, so that the method is liable to change in concentrations due to these causes. Urine serves to properly secrete excess water or a waste material for keeping homeostasis (acid-base equilibrium and the like) of body fluids, and concentrations in urine do not altogether indicate directly concentrations within the body. Thus, the method is not satisfactory either as a diagnostic method in view of sensitivity.
In the method for measuring polyamines by the Isobe et al enzymatic method, there is still room for improvement in simplicity because two types of amine oxidases derived from bacteria and fungi are used and a two-step enzymatic reaction is needed.
These methods for measuring polyamines are to measure total polyamines in urine or blood, and do not specifically measure specific polyamine compounds. Accordingly, disease conditions or physiological conditions which can be diagnosed are naturally restricted, and the methods are considered to be problematic in diagnostic precision regarding some types of disease conditions or physiological conditions.